Chondrocytes In Vitro Systems Allowing Study of OA

Osteoarthritis (OA) is an extremely complex disease, as it combines both biological-chemical and mechanical aspects, and it also involves the entire joint consisting of various types of tissues, including cartilage and bone. This paper describes the methods of conducting cell cultures aimed at searching for the mechanical causes of OA development, therapeutic solutions, and methods of preventing the disease. It presents the systems for the cultivation of cartilage cells depending on the level of their structural complexity, and taking into account the most common solutions aimed at recreating the most important factors contributing to the development of OA, that is mechanical loads. In-vitro systems used in tissue engineering to investigate the phenomena associated with OA were specified depending on the complexity and purposefulness of conducting cell cultures.

Introduction
Tissue engineering, a field of science investigating cell cultures, tissues, and organs and suggesting alternative solutions that change the approach to treatment, has experienced dynamic development in recent years. Reconstruction of damaged tissues or organs via tissue engineering is a multistep process. Initially, a tissue fragment is collected from the patient's or another donor's body. The collected specimen is processed by, for example, releasing the cells from their natural matrix. Then the resulting cells are plated on culture media. At the next stage, the cells are cultured in the medium under specific conditions and they replicate [1]. One of the fields of tissue engineering focus is the research on a civilization disease, osteoarthritis (OA). To improve the therapeutic options for this increasingly prevalent condition, scientists investigate joint building cells and analyze the effects of selected factors on the cells' behavior. As OA is a complex disease with development affected by numerous factors, in-vitro research is multifaceted and interdisciplinary. Therefore, it is extremely difficult to recreate in a laboratory all the actual conditions the body experiences in the course of the disease. However, with technologies that enable the production of modern culture carriers and innovative techniques for conducting cell cultures, it is possible to make these conditions increasingly similar to those prevailing in-vivo.
This work describes the methods of conducting cell cultures aimed at searching for the causes of OA development, therapeutic solutions, and methods of preventing the disease. It presents the systems for the cultivation of chondrocytes (cartilage cells) depending on the level of their structural complexity, and taking into account the most common solutions aimed at recreating the most important factors contributing to the development of OA, that is mechanical loads, as shown in Figure 1.

What Is Osteoarthritis
Osteoarthritis, the most common type of arthritis, is one of the most common diseases of civilization in the world [2]. Even though the problem is very common, researchers are yet to fully understand the processes behind the degeneration of joints. It is an extremely complex issue, as it combines both biological-chemical and mechanical aspects, and it also involves the entire joint consisting of various types of tissues, including cartilage and bone [3]. Degenerative changes can occur in any type of joint; however, they most often occur in the knee and hip joints in elderly people, more often in women than in men, as well as in athletes, overweight people, and people with non-physiological posture, for example, with varus or valgus of the lower extremities [4,5].
Degenerative changes in the joints include wearing and loss of articular cartilage, growth of bone spurs, and remodeling of subchondral bone. These changes are accompanied by inflammation. OA can develop in any person but the groups mentioned above are at a greater risk. Osteoarthritis is diagnosed primarily in the elderly, in whom cartilage tissue loss in the joints is observed after many years of functioning. OA causes severe pain and limits normal physical activity of the patients [6]. It is becoming a more and more frequently diagnosed disease, but the very process of its occurrence and development is still not fully understood. The degenerative changes are influenced by aspects related to mechanics, biology and chemistry, as well as the element of randomness depending on the individual characteristics of a given organism. The complexity of the disease makes it an interesting and challenging subject of research [7].

What Is Osteoarthritis
Osteoarthritis, the most common type of arthritis, is one of the most common diseases of civilization in the world [2]. Even though the problem is very common, researchers are yet to fully understand the processes behind the degeneration of joints. It is an extremely complex issue, as it combines both biological-chemical and mechanical aspects, and it also involves the entire joint consisting of various types of tissues, including cartilage and bone [3]. Degenerative changes can occur in any type of joint; however, they most often occur in the knee and hip joints in elderly people, more often in women than in men, as well as in athletes, overweight people, and people with non-physiological posture, for example, with varus or valgus of the lower extremities [4,5].
Degenerative changes in the joints include wearing and loss of articular cartilage, growth of bone spurs, and remodeling of subchondral bone. These changes are accompanied by inflammation. OA can develop in any person but the groups mentioned above are at a greater risk. Osteoarthritis is diagnosed primarily in the elderly, in whom cartilage tissue loss in the joints is observed after many years of functioning. OA causes severe pain and limits normal physical activity of the patients [6]. It is becoming a more and more frequently diagnosed disease, but the very process of its occurrence and development is still not fully understood. The degenerative changes are influenced by aspects related to mechanics, biology and chemistry, as well as the element of randomness depending on the individual characteristics of a given organism. The complexity of the disease makes it an interesting and challenging subject of research [7].
Scientific literature on the development of osteoarthritis has identified the causes and effects of OA, as listed in Table 1 [8][9][10][11][12][13][14][15][16]: Osteoarthritis develops gradually ( Figure 2). Its onset is characterized by a loss of synovial fluid between the articular surfaces covered with hyaline cartilage, which initiates inflammation. The cartilage begins to lose its absorbing properties and its continuity, microcracks appear, but its surface remains smooth. At the next stage, cartilage cells undergo gradual growth (hypertrophy), which leads to their death. This is accompanied by an increasing network of microcracks in the subchondral bone layer. In this phase of the disease, the surface of the cartilage top layer is rough and cracked, which further reduces its shock-absorbing ability. Then, as a result of thinning and delamination, local defects in the top layer of cartilage can be observed. Subsequently, cysts form in the middle layer of the cartilage and osteophytes (bone spurs) appear on the border between the bone and the cartilage. This process is associated with the growth of blood vessels from the subchondral bone into the cartilage [17]. In the final stage of the disease, the middle and basal layers of the cartilage erode. At this point, the cartilage is almost completely worn away and the bone surface is exposed. The bone spurs are now clearly visible and impede the movement of the joint. Moreover, at this stage of the disease, bone remodeling begins [15,18,19].

Cause Symptom/Effect
Previous injuries Reduction of the cushioning properties of cartilage Osteoarthritis develops gradually ( Figure 2). Its onset is characterized by a loss of synovial fluid between the articular surfaces covered with hyaline cartilage, which initiates inflammation. The cartilage begins to lose its absorbing properties and its continuity, microcracks appear, but its surface remains smooth. At the next stage, cartilage cells undergo gradual growth (hypertrophy), which leads to their death. This is accompanied by an increasing network of microcracks in the subchondral bone layer. In this phase of the disease, the surface of the cartilage top layer is rough and cracked, which further reduces its shock-absorbing ability. Then, as a result of thinning and delamination, local defects in the top layer of cartilage can be observed. Subsequently, cysts form in the middle layer of the cartilage and osteophytes (bone spurs) appear on the border between the bone and the cartilage. This process is associated with the growth of blood vessels from the subchondral bone into the cartilage [17]. In the final stage of the disease, the middle and basal layers of the cartilage erode. At this point, the cartilage is almost completely worn away and the bone surface is exposed. The bone spurs are now clearly visible and impede the movement of the joint. Moreover, at this stage of the disease, bone remodeling begins [15,18,19].  Bone spur growth is commonly known as osteophytosis. It consists in the growth of bones in the form of so-called osteophytes. During development of degenerative changes in the joint, osteophytes grow from the bone into the cartilage, mostly where the tissues and the joint capsule meet, that is on the cartilage margin ( Figure 2). However, central osteophytes [20], formed on articular surfaces, can also be observed.
It is important to remember that degenerative changes in cartilage begin at the cellular level. Chondrocytes, subjected to mechanical stresses causing their hypertrophy and apoptosis, produce an increased amount of biochemical factors, such as vascular endothelial growth factor (VEGF) [15,[21][22][23]. VEGF directly induces growth of a new blood vessel network in a process called angiogenesis. As the new blood vessels grow into the cartilage, they provide appropriate conditions for the growth of bone spurs (osteophytes) [21,[24][25][26][27].
To study the processes taking place in the cartilage in the course of osteoarthritis, researchers try to recreate in-vivo conditions in their in-vitro tests. The following sections present the in-vitro systems used in tissue engineering to investigate the complex phenomena associated with osteoarthritis.

Cell Cultures
Establishing a cell culture for in-vitro studies begins with the collection of selected cells from the body. In a living organism, cells are closely related to the surrounding spatial structures, which control their nutrition, metabolism, morphology, and degradation. It is therefore important to ensure that the growth conditions in the cell culture are as similar to those in-vivo as possible. Those conditions involve a medium of appropriate type and composition, the right temperature, or access to oxygen and carbon dioxide [28,29].
There are two types of cell cultures. The first one is called an adherent culture. It consists of a single layer of cells (monolayer), and is also known as 2D culture. This type of culture is popular, as it is relatively easy to establish and manage. However, it does not sufficiently reproduce the complex nature of reactions occurring in a living organism that is necessary in some experiments. In order to make the processes taking place in the cultures similar to those occurring in-vivo, a second type of culture can be utilized, that is 3D culture, which accounts for the spatial relationships of the living tissue. This way it is possible to more accurately reproduce the actual conditions of the body in a laboratory [30,31].
When planning an effective culture of cartilage cells, two main factors must be considered to ensure that the in-vitro culture is compatible with the in-vivo one. The first one is the relationships between the cell and its environment, i.e., both biochemical and physical aspects. Their effects on cellular response are investigated by mechanobiology [32,33]. An optimal culture environment maximally similar to in-vivo conditions in terms of temperature, access to the medium and other elements mentioned above, is necessary for the proper growth and development of the culture. The second important factor that should be analyzed when investigating the occurrence and development of osteoarthritis is the effect of mechanical stress on cartilage cells.
Each tissue is made up of cells that interact with each other and also react to external factors. Therefore, any force applied to the body causes reactions not only on the macroscopic scale, visible to the human eye, but also on the microscopic level as it affects the cells. The way the cells respond to the surrounding environment results directly from their mechanical and biochemical properties. It seems that the mechanical properties have a key impact on the strength parameters of tissues. Tissue engineering, the aim of which is to produce in a laboratory fully functional tissues that can replace damaged parts of the human body, has significantly contributed to the development of mechanobiology. In-vivo, cells receive signals from neighboring cells, react to information transmitted by enzymes, and adapt to changes in their environment. To recreate the mechanisms operating at the cellular level in laboratory conditions, it is necessary to investigate the above-mentioned factors and determine their impact on the final result. A change in cells' functioning may entice a modification of their strength parameters and, consequently, of the biochemical signals they would send to the other cells. Therefore, to establish a culture that can be reliably used to investigate the functioning of the entire organ, it is necessary to carefully analyze the cells or cell aggregates and treat them not only as a biological but also a mechanical element.
Bearing all these factors in mind, the researchers have been developing the aforementioned types of 2D and 3D cultures to provide cells with conditions as close as possible to in-vivo ones and also to make these studies as cost-effective as possible.

Monolayer Cell Cultures
Adherent (2D) cultures are usually carried out on a flat surface, most often in a Petri dish, a multi-well polystyrene plate, or a culture flask. Polystyrene is a transparent plastic material, with the type of porosity that makes cell adhesion very effective.
The greatest advantages of a 2D culture are its simplicity and high efficiency, and the possibility of producing a large culture from a small sample. Moreover, as the cells are arranged in a single layer, it is possible to provide all of them with the same amount of nutrients, which increases the probability of obtaining a stable and uniform level of cell growth and proliferation [34].
A disadvantage of this solution is its low similarity to in-vivo conditions. In a living organism, the cells receive and react to stimuli from all dimensions. The isolation of a single layer of cells from the spatial structure destroys specific natural interactions [35]. Moreover, most 2D cultures do not allow for free flow of the medium, which is yet another deviation from natural conditions, where nutrients freely reach the cells and the waste products are gradually removed [36][37][38].
Monolayer cultures of chondrocytes are quite effective in testing the effects of selected mechanical loads or the substrate. A group of US scientists investigated the effect of hydrostatic pressure on the biochemical reactions (factors such as aggrecan, collagen of type I and II, and β-actin) in chondrocytes [39]. The literature provides more examples of testing the load in monolayer cultures [40]; however, it should be remembered that to restore the proper mechanotransduction of chondrocytes, the extracellular matrix (ECM) is necessary, in which the cells freely perceive stimuli and transmit signals in space [41].

Spatial Cell Cultures
The second type of cell cultures are spatial (3D) cultures that allow for in-vitro reconstruction of a characteristic, spatial tissue system, including the ECM. Under these conditions the cells can interact with each other, respond to biochemical and biomechanical stimuli from the surrounding structures, or stimulate the expression of selected genes that ensure proper functioning of the tissues [42,43]. In a spatial culture the biochemical information is transferred between the cells and between the cells and the ECM in the same way as in the body [44]. Spatial cell cultures can have different forms. The main difference is establishing the culture with or without a scaffold.

Scaffoldless
In a scaffoldless culture the cells are forced to clump together by shaking or vortexing the culture vessel. Chondrocytes form so-called aggregates and spheroids up to 5 mm in diameter that functionally resemble a tissue [45,46]. According to one definition, an aggregate is a macromolecular compound that is an essential component of the cartilage matrix, while spheroids are spatial clusters of cells with a spherical shape, comprising the aggregates. Thanks to their spatial structure, they allow for laboratory (in-vitro) analysis of the cell-cell and cell-ECM interactions. Importantly, the compressive strength of such a cartilage-like structure is over one third of that of the actual cartilage tissue [47]. This is important information when conducting mechanical research typical of tissue engineering focused on OA. Spheroids are characterized by a layered structure, with different properties of particular zones. The inner layer, known as the necrotic core, is formed as a result of hampered diffusion of the toxic waste outside the spheroid and low availability of oxygen and nutrients. The middle layer is the area of equilibrium between the nutrients supplied to the inside and the toxins removed outside. The outer zone of the spheroid is the area where cell proliferation is very intense. This is due to the unlimited access to oxygen and nutrients, and the ease of removing harmful metabolites. Spheroids are used for works analyzing cell metabolism, cell proliferation and differentiation, intercellular interactions, ECM synthesis, and vascularization of cell clusters. A disadvantage of this solution is short viability of cells due to the central necrosis resulting from impaired access of nutrients. Cartilage-like spheroids can also be obtained by growing cells in a hanging drop [48]. However, the scaffoldless cultures have a less organized structure than the cartilage tissue in a living organism [49].

Scaffolds
In in-vivo cultures, the cells are properly arranged in space thanks to the ECM, which not only provides cells with an appropriate scaffold, but also regulates their reproduction and differentiation. To recreate an in-vivo culture in laboratory conditions, it is necessary to use a matrix substitute made of appropriate materials and fulfilling strictly defined functions. Scaffolds solve the problem of possible necrosis in the central part of the culture by providing a structure with parameters that allow free flow of the medium with nutrients or information in the form of proteins. The porous structure enables the nourishment of cells embedded in the inner pores of the scaffold (Figure 3) [1].
ygen and nutrients. The middle layer is the area of equilibrium between the nutrients supplied to the inside and the toxins removed outside. The outer zone of the spheroid is the area where cell proliferation is very intense. This is due to the unlimited access to oxygen and nutrients, and the ease of removing harmful metabolites. Spheroids are used for works analyzing cell metabolism, cell proliferation and differentiation, intercellular interactions, ECM synthesis, and vascularization of cell clusters. A disadvantage of this solution is short viability of cells due to the central necrosis resulting from impaired access of nutrients. Cartilage-like spheroids can also be obtained by growing cells in a hanging drop [48]. However, the scaffoldless cultures have a less organized structure than the cartilage tissue in a living organism [49].

Scaffolds
In in-vivo cultures, the cells are properly arranged in space thanks to the ECM, which not only provides cells with an appropriate scaffold, but also regulates their reproduction and differentiation. To recreate an in-vivo culture in laboratory conditions, it is necessary to use a matrix substitute made of appropriate materials and fulfilling strictly defined functions. Scaffolds solve the problem of possible necrosis in the central part of the culture by providing a structure with parameters that allow free flow of the medium with nutrients or information in the form of proteins. The porous structure enables the nourishment of cells embedded in the inner pores of the scaffold (Figure 3) [1]. Scaffolds should be made of materials ensuring appropriate porosity and permeability. In addition, the material from which the culture scaffold is made must be at least biocompatible, that is must have appropriate surface roughness that allows for cell deposition on its surface. A proper scaffold should have mechanical properties close enough to those of the cartilage to allow the sample with cells to be subjected to loads similar to actual ones [50]. The loads to which cells respond when the tissue is not yet damaged are in the range of 6 to 20 MPa [51]. Depending on the material used to make the scaffolds, they can be divided into natural, synthetic, and composite ones.
Considering the above information, the most important functions of the cell culture scaffold are as follows: • Creating a spatial support for the cultured cells, • Ensuring proper adhesion of the cells to the substrate, • Enabling development and proliferation of cells through appropriate geometric properties, Scaffolds should be made of materials ensuring appropriate porosity and permeability. In addition, the material from which the culture scaffold is made must be at least biocompatible, that is must have appropriate surface roughness that allows for cell deposition on its surface. A proper scaffold should have mechanical properties close enough to those of the cartilage to allow the sample with cells to be subjected to loads similar to actual ones [50]. The loads to which cells respond when the tissue is not yet damaged are in the range of 6 to 20 MPa [51]. Depending on the material used to make the scaffolds, they can be divided into natural, synthetic, and composite ones.
Considering the above information, the most important functions of the cell culture scaffold are as follows: • Creating a spatial support for the cultured cells, • Ensuring proper adhesion of the cells to the substrate, • Enabling development and proliferation of cells through appropriate geometric properties, • Ensuring living tissue-like mechanical properties of the entire culture structure.
An additional advantage of the scaffold-based culture is the possibility of testing the cell response to various factors and controlling their response by incorporating specific elements into the scaffold structure. These can be for example bioactive, growth factor-rich layers coated on the surface of the scaffolds, as described in detail by Bjelić and Finšgar in 2021 [52].
In order for the scaffold to have the expected functions, it is also necessary to take into account additional features of the material from which the scaffold is built. The material should or can have the following properties: • Biocompatibility-the material cannot be toxic and cannot cause an immune system reaction, • Biodegradability-the scaffold material may degrade over time and under certain conditions, • Specific surface properties-the scaffold material must have an appropriate structure ensuring cell adhesion to the substrate, • Porosity-the material must ensure the possibility of growing a large number of cells with a relatively small volume of the substrate, and must allow for free migration of cells and even supply and removal of nutrients, • Appropriate mechanical properties-the material should meet the specific requirements for a given type of culture.
Accordingly, the materials used to build the cell culture scaffold may be, as indicated above, of natural, synthetic, or composite origin.
Natural scaffolds are mostly based on collagen and hyaluronic acid (hyaluronan/hyaluronic acid), which are found in natural cartilage tissue. Due to their origin, these scaffolds are recognized by cartilage cells and significantly influence the course of the culture, for example, by supporting cell proliferation and building of the cartilage tissue. Collagen, as a protein found in the human body, including the intercellular fluid, skin, tendons, hair or nails, meets the requirements for biocompatibility.
Moreover, collagen is also a component of connective tissue and cell matrix [53]. However, it is necessary to remember the often insufficient strength properties of materials based on collagen or hyaluronic acid, and the fact that scaffolds made of natural compounds lose their strength and structural properties over time [54].
In order to obtain selected, optimal mechanical properties of the scaffolds, some studies focused on degenerative changes utilize scaffolds made of natural substances derived from outside the human body, such as alginates obtained from seaweed or silk characterized by a fibrous structure [55,56]. Materials based on silk may have diverse structural forms such as fibrous, porous or thin film. The properties such as their versatility, biodegradation, and biocompatibility as well as favorable capability of strengthening attachment, proliferation, and differentiation of chondrocytes are crucial to applying this kind of scaffold in tissue engineering [57,58].
Synthetic scaffolds allow for customization of physical and chemical properties during their manufacture. Thanks to their appropriate features and biodegradability, synthetic polymers are increasingly used in tissue engineering. The most popular synthetic polymers used for the production of culture scaffolds are polylactide (PLA), polyglycolide (PGA), and polycaprolactone (PCL). A very important property of synthetic polymers is their biodegradability, that is gradual degradation of the material into substances that are safe for the body, such as carbon dioxide and minerals. The time of biodegradation is controlled and depends on the type of culture and, inter alia, on the diameter of the fibers. Current technology allows for programming selected material or biochemical properties depending on the chemical composition of the raw material. An alternative to collagen is an elastinlike polymer. Elastin is present in the human body as a component of connective tissue, intercellular matrix, tendons, ligaments, skin, and walls of large blood vessels. The scaffolds made of elastin-like polypeptides feature properties similar to those of elastin, primarily flexibility [59]. These properties are also helpful in research involving the cultures of fibroblasts, the skin-building cells [50].
An interesting solution in this field is the use of composite scaffolds. Design of the composite material for a scaffold for cell culture requires the appropriate selection of materials for the matrix and for the modifying phase, with biocompatibility being the basic, decisive criterion. Modifying phases can have the form of a powder, porous structures and solid, dense materials, while the matrix is made, for example, from fibers [60] (Figure 4).
Tissue engineering applied in studies on osteoarthritis-related factors and cartilage repair increasingly use scaffolds printed on bioprinters, where cells are seeded onto consecutive layers of the print [61,62]. the cultures of fibroblasts, the skin-building cells [50].
An interesting solution in this field is the use of composite scaffolds. Design of the composite material for a scaffold for cell culture requires the appropriate selection of materials for the matrix and for the modifying phase, with biocompatibility being the basic, decisive criterion. Modifying phases can have the form of a powder, porous structures and solid, dense materials, while the matrix is made, for example, from fibers [60] (Figure  4). Tissue engineering applied in studies on osteoarthritis-related factors and cartilage repair increasingly use scaffolds printed on bioprinters, where cells are seeded onto consecutive layers of the print [61,62].
To make the culture scaffolds as similar as possible to the structure of cartilage, they can be produced as hydrogels, nanofibers, foam or sponge [1]. These structures are porous and permeable, but the production of an uncontrolled structure can be a limitation. The technology of bioprinting may provide a solution to the problem of the lack of control over the structural properties, as it enables planning the geometry of the hydrogel structure and simultaneous seeding of cells [63]. The hydrogels used in this technology can be made of both natural and synthetic polymers. By selecting polymers and combining them in various proportions, it is possible to obtain hydrogels with different mechanical properties, tailored for a given research task [64].
A cell culture, based on a hydrogel structure, begins with combining the cells with a liquid pre-hydrogel, which is then exposed to external factors such as, for example, radiation of a given wavelength, temperature, or appropriate chemicals. When exposed to the selected factor, the polymer begins to gel and swell [65], resulting in the formation of the expected structure intended for the cell culture.

Explants
Research in an isolated tissue fragment called an explant, is another solution employed in tissue engineering [37]. When conducting research in cell cultures, it is important to ensure conditions that guarantee the development of the research material imitating the investigated organ. This material can perform the same functions as in-vivo tissue. To make the culture scaffolds as similar as possible to the structure of cartilage, they can be produced as hydrogels, nanofibers, foam or sponge [1]. These structures are porous and permeable, but the production of an uncontrolled structure can be a limitation. The technology of bioprinting may provide a solution to the problem of the lack of control over the structural properties, as it enables planning the geometry of the hydrogel structure and simultaneous seeding of cells [63]. The hydrogels used in this technology can be made of both natural and synthetic polymers. By selecting polymers and combining them in various proportions, it is possible to obtain hydrogels with different mechanical properties, tailored for a given research task [64].
A cell culture, based on a hydrogel structure, begins with combining the cells with a liquid pre-hydrogel, which is then exposed to external factors such as, for example, radiation of a given wavelength, temperature, or appropriate chemicals. When exposed to the selected factor, the polymer begins to gel and swell [65], resulting in the formation of the expected structure intended for the cell culture.

Explants
Research in an isolated tissue fragment called an explant, is another solution employed in tissue engineering [37]. When conducting research in cell cultures, it is important to ensure conditions that guarantee the development of the research material imitating the investigated organ. This material can perform the same functions as in-vivo tissue.
The simplest solution is to isolate a fragment of a living tissue. This allows for reducing costs and simplifying the research material production, and provides all the structural conditions and mechanical properties of a culture so important in OA-oriented research [66]. Unfortunately, obtaining explants can be very difficult due to the procedures and availability of explants derived from living organisms. The advantage of explants is not only the fact that they do not grow, but also that they maintain the functionality of cells which, reacting to external stimuli, are consequently capable, for example, of releasing specific biochemical factors. A disadvantage of such a culture is its short viability (up to 10 days) [56].

Decellularized Composite Scaffolds
A very interesting type of scaffolds are hybrid scaffolds which incorporate decellularized matrix with synthetic biomaterials. Generally, decellularization is utilized to remove cellular components from articular cartilage. It is needed to preserve structural proteins and other molecules during the process of decellularization. Gel created from this decellularized ECM is mechanically more similar to the native cartilage tissue and capable of inducing chondrogenesis. Once a decellularized ECM is prepared, it can be coated onto culture dish or multi-hole plates as well as onto a scaffold prepared for instance by 3D prototype printing techniques. Application of this type of scaffold is very promising in regeneration tissue study [67][68][69][70].

Bioreactors
In in-vivo models, nutrients and body fluids are delivered to the cells continuously rather than intermittently. Therefore, even many three-dimensional models of cell cultures, e.g., spheroids, in which the medium is administered to the cells from time to time, do not fully reflect the natural conditions. In-vitro cultures that enable continuity of physiological processes and provide control over the supplied compounds can be grown in bioreactors. These complex systems are usually used for spatial cultures but, depending on the needs, monolayer cell cultures can also be grown this way [71]. Due to their complexity, these systems are often called bioreactors.
Cell cultures require constant monitoring of numerous parameters, i.e., temperature, pH, access to nutrients and oxygen, as well as other physical and chemical parameters depending on the purpose of the culture. A bioreactor is a device that facilitates the control of all factors influencing the final effect. There are many literature definitions of bioreactors: "A bioreactor is the general term applied to a closed culture environment, that is usually mixed, that enables control of one or more environmental or operating variables that affect biological processes" [72], "The role of a bioreactor is to mimic the in-vivo conditions of a tissue. The bioreactor should be able to control the different variables that define the environment of the tissue" [73], "Bioreactors are devices in which biological or biochemical processes develop under a closely monitored and tightly controlled environment" [74]. These definitions clearly show that bioreactors used in tissue engineering can be defined as systems that enable cell cultures to be carried out in conditions similar to natural ones and in which the monitoring of variable biomechanical parameters is possible. Depending on the type of culture, the bioreactors can have various structural and technical parameters.
The main advantage of bioreactor cultures is the possibility of providing the cells with specific substances or other factors, and then monitoring their effect on the cell culture. In the case of cartilage engineering, mechanical loads are an important factor influencing the development of the cultures. This is due to the fact that in a living organism, chondrocytes are subjected to steady and cyclical loads caused by body movements. Therefore, in order to best reproduce in-vivo conditions in the laboratory, it is worth using a bioreactor that enables not only the introduction of additional loads, but also controlling their impact on the cells [75].
Tissue engineering was used in research on the possibility of in-vitro production of a cartilage-like structure that meets the mechanical requirements for a natural tissue and its implantation in a patient. Implementation of this purpose required three key elements: autologous cartilage cells (chondrocytes), a scaffold on which the cells can be placed, and a bioreactor, i.e., a device reproducing in-vivo culture conditions.

Types of Bioreactors for Chondrocyte Culture
Depending on the type of load to which the culture is subjected, the literature distinguishes several basic designs of bioreactors. Considering how joints, and specifically, cartilage tissue, work and how mechanical loads affect the development of osteoarthritis, apart from controlling biophysical factors, the most important aspects seem to be shear force and compression. Shear load bioreactors can implement the load by shear contact [76] or fluid flow [77], for example, in so-called rotating wall vessel (RWV) bioreactors [78]. Pressure bioreactors, on the other hand, can be divided into indirect pressure systems ( Figure 5), in which hydrostatic pressure is generated [79], and direct pressure systems using various types of pistons [80]. tilage tissue, work and how mechanical loads affect the development of osteoarthritis, apart from controlling biophysical factors, the most important aspects seem to be shear force and compression. Shear load bioreactors can implement the load by shear contact [76] or fluid flow [77], for example, in so-called rotating wall vessel (RWV) bioreactors [78]. Pressure bioreactors, on the other hand, can be divided into indirect pressure systems ( Figure 5), in which hydrostatic pressure is generated [79], and direct pressure systems using various types of pistons [80]. Indirect pressure bioreactors have a piston-like element that creates hydrostatic pressure by compressing the gas that transfers the load to the medium surrounding the cell culture or by squeezing the medium. In direct pressure bioreactors the piston compresses the culture fluid directly, bypassing the gaseous layer [81].
Shear bioreactors provide a friction load as a result of contact shear force between the working surface and the culture structure or hydrodynamic shear in the form of the fluid flow shear [82]. The first type of direct shear bioreactors is equipped with a surface moving over the culture structure [83]. The second type of bioreactors are, for example, RWV ones, in which the outer cylinder making up the bioreactor chamber rotates and causes the fluid to vortex. Spinner Flask systems provide another example, in which the fluid movement is triggered by a rotating bar placed at the bottom of the chamber [84]. A solution that can be used to generate friction load is perfusion of a spatial, porous culture structure in so-called perfusion systems. Fluid flow control is often enforced by peristaltic pumps that ensure a linear flow velocity of the medium [85,86].
The RWV-type bioreactor is considered the best device for the culture of cartilage cells due to the optimized method of delivering nutrients [87]. Various variants of this construction are employed, for example rotation-based bioreactor systems [88]. The rotating part is the base of the cylinder, and the scaffolds can be freely suspended in the medium or attached to the curved walls of the bioreactor. The culture can also be seeded on Indirect pressure bioreactors have a piston-like element that creates hydrostatic pressure by compressing the gas that transfers the load to the medium surrounding the cell culture or by squeezing the medium. In direct pressure bioreactors the piston compresses the culture fluid directly, bypassing the gaseous layer [81].
Shear bioreactors provide a friction load as a result of contact shear force between the working surface and the culture structure or hydrodynamic shear in the form of the fluid flow shear [82]. The first type of direct shear bioreactors is equipped with a surface moving over the culture structure [83]. The second type of bioreactors are, for example, RWV ones, in which the outer cylinder making up the bioreactor chamber rotates and causes the fluid to vortex. Spinner Flask systems provide another example, in which the fluid movement is triggered by a rotating bar placed at the bottom of the chamber [84]. A solution that can be used to generate friction load is perfusion of a spatial, porous culture structure in so-called perfusion systems. Fluid flow control is often enforced by peristaltic pumps that ensure a linear flow velocity of the medium [85,86].
The RWV-type bioreactor is considered the best device for the culture of cartilage cells due to the optimized method of delivering nutrients [87]. Various variants of this construction are employed, for example rotation-based bioreactor systems [88]. The rotating part is the base of the cylinder, and the scaffolds can be freely suspended in the medium or attached to the curved walls of the bioreactor. The culture can also be seeded on cylindrical elements attached to the central shaft connected to the rotating base of the cylinder ( Figure 6).  Research results published in 1996 demonstrated that the Spinner Flask bioreactor (see Figure 7) allowed for obtaining chondrocytes of a very regular shape, and the number of proliferated cells was up to 70% greater than in static cultures [89]. Research results published in 1996 demonstrated that the Spinner Flask bioreactor (see Figure 7) allowed for obtaining chondrocytes of a very regular shape, and the number of proliferated cells was up to 70% greater than in static cultures [89]. Research results published in 1996 demonstrated that the Spinner Flask bioreactor (see Figure 7) allowed for obtaining chondrocytes of a very regular shape, and the number of proliferated cells was up to 70% greater than in static cultures [89]. Direct friction bioreactors are equipped with an element whose surface moves on the surface of the cell culture with a light axial pressure. This movement can cause sliding friction or rolling resistance as shown in the exemplary design of the structure in Figure 8 [76,90]. The friction comes from the ceramic hip ball that makes cyclic movements compressing the scaffold with simultaneous rotation, which causes friction on the culture surface (combination of compression and shear forces). Direct friction bioreactors are equipped with an element whose surface moves on the surface of the cell culture with a light axial pressure. This movement can cause sliding friction or rolling resistance as shown in the exemplary design of the structure in Figure 8 [76,90]. The friction comes from the ceramic hip ball that makes cyclic movements compressing the scaffold with simultaneous rotation, which causes friction on the culture surface (combination of compression and shear forces).
Research results published in 1996 demonstrated that the Spinner Flask bi (see Figure 7) allowed for obtaining chondrocytes of a very regular shape, and the of proliferated cells was up to 70% greater than in static cultures [89]. Direct friction bioreactors are equipped with an element whose surface move surface of the cell culture with a light axial pressure. This movement can cause friction or rolling resistance as shown in the exemplary design of the structure in [76,90]. The friction comes from the ceramic hip ball that makes cyclic movemen pressing the scaffold with simultaneous rotation, which causes friction on the cul face (combination of compression and shear forces). Flow perfusion bioreactors are equipped with a pump that forces the flow of the medium through the culture structure and controls its velocity. In addition, the cells must be seeded on porous and permeable scaffolds to allow the medium to flow inside the structure. Thanks to this and the ability to control selected factors, including the flow velocity of the medium, the cells are provided with a continuous supply of nutrients with simultaneous tangential loading, as in natural cartilage.
A combination of a flow perfusion bioreactor and a scaffold of a porous structure make it possible for the medium to reach all cells of the culture. An example of this system is a design proposed by German researchers in 2012 [91] and presented in Figure 9. With appropriate regulation of the fluid flow velocity, it is possible to obtain a tissue with a homogeneous structure and biomechanical properties corresponding to natural cartilage [92].
A combination of a flow perfusion bioreactor and a scaffold of a porous structure make it possible for the medium to reach all cells of the culture. An example of this system is a design proposed by German researchers in 2012 [91] and presented in Figure 9. With appropriate regulation of the fluid flow velocity, it is possible to obtain a tissue with a homogeneous structure and biomechanical properties corresponding to natural cartilage [92].

Lab-on-Chip Systems
Another solution bringing the in-vitro systems closer to the in-vivo ones is the use of the modern method of flow microsystems, also known as lab-on-chip (LoC). It is a microlaboratory system, built of many properly arranged elements and forming an in-vitro microplatform resembling an in-vivo culture. The LoC microsystems are equipped with cell culture sites, microchannels supplying the cells and a whole network of microstructures, through which it is possible to constantly supply the medium and control selected biomechanical factors, as in Figure 10

Lab-on-Chip Systems
Another solution bringing the in-vitro systems closer to the in-vivo ones is the use of the modern method of flow microsystems, also known as lab-on-chip (LoC). It is a microlaboratory system, built of many properly arranged elements and forming an in-vitro microplatform resembling an in-vivo culture. The LoC microsystems are equipped with cell culture sites, microchannels supplying the cells and a whole network of microstructures, through which it is possible to constantly supply the medium and control selected biomechanical factors, as in Figure 10 [93]. make it possible for the medium to reach all cells of the culture. An example of this system is a design proposed by German researchers in 2012 [91] and presented in Figure 9. With appropriate regulation of the fluid flow velocity, it is possible to obtain a tissue with a homogeneous structure and biomechanical properties corresponding to natural cartilage [92].

Lab-on-Chip Systems
Another solution bringing the in-vitro systems closer to the in-vivo ones is the use of the modern method of flow microsystems, also known as lab-on-chip (LoC). It is a microlaboratory system, built of many properly arranged elements and forming an in-vitro microplatform resembling an in-vivo culture. The LoC microsystems are equipped with cell culture sites, microchannels supplying the cells and a whole network of microstructures, through which it is possible to constantly supply the medium and control selected biomechanical factors, as in Figure 10 [93]. Figure 10. An example of a lab-on-chip system consisting of a lower and upper body part, medium supply channels and a culture channel. Reprinted with permission from [94]. Copyright 2019, Nature Biomedical Engineering.
As in bioreactors, cell cultures in lab-on-chip systems are provided with a continuous flow of the medium through a flat or three-dimensional culture structure. Additionally, the lab-on-chips enables studies on the interaction not only between different types of tissues but also between individual organs [95][96][97]. Moreover, these complex systems go beyond 2D culture research. Depending on the design of the chip, it is also possible to conduct experiments on three-dimensional cultures produced, for example, by bioprinting [98]. The flow platforms make it also possible to run co-cultures, and thus enable the analysis of the interactions of various cell lines forming tissues, organs, or whole organisms [94]. An example of such an extensive lab-on-chip system, taking into account cell cultures present in various joint tissues, is presented in [99].
The above example of a joint-on-chip platform illustrates just how complex and multilayered a lab-on-chip architecture can be. However, it should be remembered that the more complex the culture systems are, the more difficult they are to control, and the more expensive it is to produce such carriers.

Conclusions
The aim of tissue engineering is to search for solutions for conducting cell cultures that enable to study the processes taking place at the cellular level, to be able to affect them in a controlled way, and finally to propose new treatment methods. Depending on the structural complexity of the culture carriers and the investigated problem, research can be carried out in simple, monolayer, individual cell cultures or more complex systems of 3D cultures. It seems that, due to the etiology of the degenerative disease, research on the processes accompanying OA should be aimed at lab-on-chip systems; however, when expanding the culture systems one should always bear in mind that the greater their complexity, the higher the costs of their production and of conducting the research. Nevertheless, it can be assumed that the cell culture platforms used for OA research will be developed primarily in the field of material technologies and structures enabling co-cultures. The need for new methods of treating OA forces the development of tissue engineering and modern culture scaffolds enabling the possible implantation of new tissue [100]. In addition to innovative culture biomaterials, an interesting field of research is also designing new structures of bioreactors and flow microsystems that would enable the analysis of individual stages of osteoarthritis development.


Quantitative glycoproteomics analysis identifies novel FUT8 targets and signaling networks critical for breast cancer cell invasiveness

Background We recently showed that fucosyltransferase 8 (FUT8)-mediated core fucosylation of transforming growth factor-β receptor enhances its signaling and promotes breast cancer invasion and metastasis. However, the complete FUT8 target glycoproteins and their downstream signaling networks critical for breast cancer progression remain largely unknown. Method We performed quantitative glycoproteomics with two highly invasive breast cancer cell lines to unravel a comprehensive list of core-fucosylated glycoproteins by comparison to parental wild-type and FUT8-knockout counterpart cells. In addition, ingenuity pathway analysis (IPA) was performed to highlight the most enriched biological functions and signaling pathways mediated by FUT8 targets. Novel FUT8 target glycoproteins with biological interest were functionally studied and validated by using LCA (Lens culinaris agglutinin) blotting and LC–MS/MS (liquid chromatography–tandem mass spectrometry) analysis. Results Loss-of-function studies demonstrated that FUT8 knockout suppressed the invasiveness of highly aggressive breast carcinoma cells. Quantitative glycoproteomics identified 140 common target glycoproteins. Ingenuity pathway analysis (IPA) of these target proteins gave a global and novel perspective on signaling networks essential for breast cancer cell migration and invasion. In addition, we showed that core fucosylation of integrin αvβ5 or IL6ST might be crucial for breast cancer cell adhesion to vitronectin or enhanced cellular signaling to interleukin 6 and oncostatin M, two cytokines implicated in the breast cancer epithelial–mesenchymal transition and metastasis. Conclusions Our report reveals a comprehensive list of core-fucosylated target proteins and provides novel insights into signaling networks crucial for breast cancer progression. These findings will assist in deciphering the complex molecular mechanisms and developing diagnostic or therapeutic approaches targeting these signaling pathways in breast cancer metastasis. Supplementary Information The online version contains supplementary material available at 10.1186/s13058-022-01513-3.

covalent attachment of oligosaccharide chains to proteins or lipids. The carbohydrate structures of cell-surface glycoconjugates play an important role in many physiological and pathological events, including cell growth, differentiation, and transformation [3].
Fucosylation is one of the most important types of glycosylation in cancers. Altered fucosylation has been implicated in malignant transformation, invasion, and metastasis of certain types of cancers, such as hepatocellular, gastric, pancreatic, prostate, and colorectal cancers [4][5][6][7][8]. To date, 13 different fucosyltransferases (FUTs) have been identified in the human genome [9]. FUT8 is the only fucosyltransferase involved in core fucosylation (addition of fucose in α-1,6-linkage to the innermost N-acetyl glucosamine of N-glycans) [10]. Our recent study showed that upregulated FUT8 during transforming growth factor (TGF)-β-induced epithelial-mesenchymal transition (EMT) represents a novel, vicious feed-forward loop linking core fucosylation and TGF-β receptor signaling to promote EMT and breast cancer progression [11]. Besides TGF-β receptors, other FUT8 targets such as epidermal growth factor receptor and an adhesion receptor complex, α3β1 integrin, may potentiate ligand binding ability and enhance downstream signal pathways to support tumor growth and metastasis [12,13]. The pathological processes are a combination of complicated interactions of protein networks. Therefore, we need to identify additional FUT8 target proteins and their downstream signaling during breast cancer progression.
In this study, we used the comparative glycoproteomics to facilitate the identification of 392 corefucosylated glycoproteins from two highly invasive breast cancer cell lines (MDA-MB-231 and Hs578T); 140 proteins were commonly shared targets and more than 90% were membrane glycoproteins. Ingenuity Pathway Analysis revealed that these target proteins form complex signaling networks involved in controlling downstream cellular functions including proliferation, adhesion, migration, and invasion of breast cancer cells. In addition, we verified integrin αvβ5 and interleukin 6 signal transducer (IL6ST) as novel glycoprotein targets of FUT8. Core fucosylation of these molecules played important functions in breast cancer cell adhesion to vitronectin and their responsiveness to IL-6 or oncostatin M (OSM) signaling. Further understanding the signal pathways and biological effects mediated by these core-fucosylated glycoproteins could help in deciphering the complex molecular mechanisms in breast cancer metastasis and lead to more effective diagnostic or therapeutic interventions.

Immunoprecipitation, western blot analysis, and lectin blot analysis
Two days after transfection, cell lysates were clarified by centrifugation at 10,000×g for 20 min at 4 °C. Samples were incubated with indicated antibody and 50% (v/v) Protein A-agarose (Thermo Fisher Scientific, Waltham, MA) for 2 h with gentle rocking at 4 °C. Precipitated complexes were solubilized by boiling in Laemmli sample buffer, fractionated by SDS-PAGE, and transferred onto PVDF membranes, which were blocked with phosphate buffered saline (PBS; pH 7.5) containing 0.1% gelatin and 0.05% Tween 20 and blotted with the indicated antibodies. After washes, blots were incubated with horseradish peroxidase-conjugated goat anti-mouse/rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h. Reactive bands were visualized by using the VisGlow chemiluminescent substrate, horseradish peroxidase system (Visual Protein, Taiwan). For lectin blot analysis, membranes were blocked with PBS (pH 7.5) containing 0.1% gelatin and 0.05% Tween 20, detected with biotinylated Lens culinaris agglutinin (LCA) lectin (Vector Laboratories, Burlingame, CA), then incubated with horseradish peroxidase-conjugated streptavidin (Vector Laboratories).

Flow cytometry
Cells were collected and suspended in PBS, 2% FBS in a volume of 0.5 ml. Cell suspensions were incubated with fluorescein-labeled LCA (Vector Laboratories) on ice for 1 h. After a washing with ice-cold PBS three times, the cells were resuspended in 0.5 ml of PBS, 2% FBS. Flow cytometry involved use of FACSCalibur (BD Biosciences, San Jose, CA).

CRISPR/Cas9-mediated genome editing
To generate FUT8-knockout (FUT8-KO) cells with the CRISPR/Cas9 system, guide RNAs (gRNAs) targeting the human FUT8 gene at exon 3 or 6 were cloned into the GeneArt CRISPR Nuclease Vector (Thermo Fisher Scientific). After sequence verification of the insert, the CRISPR/Cas9 plasmids were transfected into MDA-MB-231 or Hs578T cells. Two days after transfection, cells underwent flow cytometry-based sorting of crRNA-expressing cell populations with orange fluorescent protein expression. These crRNA-expressing cell populations were further cultured for 1 week, and FUT8-KO cells were selected by FACS analysis with LCA binding. Genomic indel modification of FUT8 in single-cell clones was assessed by PCR and sequencing.

RNA extraction, cDNA synthesis, and RT-PCR
Total RNA was obtained from cultured cells by the TRIzol method (Thermo Fisher Scientific). First-strand cDNA synthesis with SuperScript II reverse transcriptase (Thermo Fisher Scientific) involved 5 μg RNA. The firststrand cDNA reaction was used as a template for each PCR using 2× SuperRed PCR Master Mix (BIOTOOLS, Taiwan).

Cell migration and invasion assay
Cell migration and invasion was measured in a Boyden chamber system as described [11]. For migration assays, cells (1 × 10 5 ) were placed in the upper chamber with non-coated membrane (24-well insert; 8-μm pore size; Corning Inc., Corning, NY). For invasion assays, cells (1 × 10 5 ) were placed in the top chamber with Matrigelcoated membrane (24-well insert; 8-μm pore size; Corning Inc.). The total number of cells that migrated into the lower chamber was counted after 16 h of incubation at 37 °C with 5% CO 2 . Cells that migrated to the lower surface of the filter were stained with 0.5% crystal violet, examined by bright field microscopy, and photographed. Crystal violet was then dissolved with ethanol, and absorbance was read at 570 nm. Values for migration/ invasion are expressed as the average number of OD570 per assay.

Cell-based reporter assay
Cell-based IL-6 measurement was initially performed according to the manufacturer's protocol (InvivoGen, San Diego, CA). Briefly, HEK-Blue cells were seeded at 50,000 cells per well into black clear-bottomed 96-well plates in 180 µL DMEM with 10% FBS, then incubated for 24 h with 20 µL IL-6 sample or control. A 20-µL amount of the cell culture medium was then transferred to wells of a 96-well plate containing 180 µL of QUANTI-Blue detection medium (InvivoGen) and incubated for 1-3 h. Color change indicative of secreted embryonic alkaline phosphatase (SEAP) levels was determined by using a plate reader at 620-655 nm.

SILAC-based glycoproteomic analysis of core fucosylated proteins
Parental MDA-MB-231 or Hs578T cells were cultured in 'light' SILAC media (12C6 14N2 l-Lysine and 12C6 14N4 l-Arginine; Lys0 and Arg0), and two independent FUT8-KO clones of each cell line were grown in 'medium' SILAC media (4,4,5,5-D4 l-Lysine and 13C6 14N4 l-Arginine; Lys4 and Arg6) or in 'heavy' SILAC media (13C6 15N2 l-Lysine and 12C6 15N4 l-Arginine; Lys8 and Arg10) (Thermo Fisher Scientific). After metabolic labeling, cell lysates were extracted by using RIPA buffer. The core fucosylated glycoproteins were enriched with LCA-agarose beads (Vector Laboratories). After washing with PBS, on-beads trypsin digestion followed by the N-deglycosylation of N-glycosidase F released N-linked deglycosylated peptides from the resin. Finally, the released peptides were desalted via C18 Ziptip (Millipore) before mass spectrometry [14]. The deglycosylated peptides were analyzed by using a reverse phase NanoLC/MS/MS system consisting of NanoUPLC (NanoACQUITY, Waters, Milford, MA) and a high-resolution mass spectrometer (Orbitrap Elite, Thermo Fisher Scientific). Protein identification and relative abundance quantification involved using Proteome Discoverer (v 1.4.1.14, Thermo Fisher Scientific). The MS/MS spectra were searched with the Mascot engine (v2.5, Matrix Science, Boston, MA) against the SwissProt Homo sapiens (Human) database. Quantification node (Precursor Ions Quantifier) was used for calculation of SILAC ratio for each peptide pair and enabled the ratio calculation by replacing missing quantification values with minimum intensity.

Pathway analysis of FUT8 target glycoproteins
Ingenuity Pathway Analysis (IPA) software (QIAGEN) was used to investigate the pathways that FUT8 targeting glycoproteins in invasive breast cancer cells may be involved in. To fulfill the format requirement of IPA, the average ratio of the intensity of heavy/light and medium/ light for each target FUT8 target protein identified in MDA-MB-231 or Hs578T cells was calculated and transformed into fold change (= − 1/average ratio). When the data were imported into IPA, the gene name provided by proteomics analysis was assigned as the ID and mapped by Gene Symbol; the transformed fold change was assigned as Expression Fold Change. Individual datasets underwent core analysis including enrichment of canonical pathways, diseases, functions, upstream regulators, and networks. Enrichment of diseases and functions was summarized into categories based on molecular and cellular functions and ranked by p value.
Similar data transformation and core analysis was performed for the 140 FUT8 target proteins commonly identified in the two cell lines. Upstream regulator analysis is a tool that analyzes connections to target proteins via coordinator expression to identify potential upstream regulators including transcription factors and any gene or small molecule that was observed experimentally to affect gene expression in direct or indirect ways [15]. The Z score determines whether an upstream regulator has significantly more 'activated' predictions than 'inhibited' predictions (z > 0) or vice versa (z < 0). Six regulators showing activation z score > 2 or < − 2 and thus predicted to be significantly activated or inhibited in cells with FUT8 knockout were selected for constructing pathways linking FUT8 target proteins to functional outcomes. With the FUT8 target proteins as mediators, the pathways regulated by FUT8 core fucosylation and responsible for phenotypical outcomes were revealed.

Identification of N-glycopeptides
Purified proteins were reduced with 10 mM dithiothreitol at 60 °C for 1 h, alkylated with 50 mM iodoacetamide in 25 mM ammonium bicarbonate buffer for 45 min in the dark at room temperature, then treated overnight with sequencing grade trypsin at an enzyme-to-substrate ratio of 1:50 at 37 °C. The digested products were then diluted with formic acid to a final concentration with 0.1% and further cleaned up by using ZipTip C18 (Millipore) before LC-MS/MS analysis. The peptide mixture was analyzed by nanoLC-MS/MS on a Tribrid Mass Spectrometer (Orbitrap Fusion Lumos, Thermo Fisher Scientific) coupled to an Easy-nLC 1200 System (Thermo Fisher Scientific). An HCD-product dependent-EThcD (HCD-pd-EThcD) workflow was used to additionally trigger EThcD events upon detecting signature glycan oxonium ions (m/z 204.0867, 138.0545, and 366.1396) during HCD fragmentation [16]. For glycopeptide identification, the MS raw data were searched by using the Byonic algorithm (v2.16.11, Protein Metrics). Glycan was searched with the default mammalian N-glycan database. To ensure high data quality, only glycopeptides with Byonic score > 200 were used for further analysis. The most likely glycan was predicted by using GlycoWorkbench (v2.1) from the glycan mass of the Byonic result, and the identified core-fucosylated glycopeptides were further confirmed by manual data inspection [17].

Statistical analysis
Data are expressed as mean ± SD and were analyzed by paired t test. p < 0.05 was considered statistically significant.

FUT8 knockout (KO) suppresses migration and invasiveness of MDA-MB-231 or Hs578T breast carcinoma cells
FUT8 is overexpressed in many human cancers and has been found associated with multiple cancer-related processes [5,10,14,16,17]. Our previous study showed that upregulated FUT8 during the TGF-β-promoted epithelial-mesenchymal transition (EMT) represents a pathological feed-forward regulatory mechanism linking core fucosylation with TGF-β receptor signaling to accelerate the EMT and promote breast cancer metastasis [11]. To search for additional FUT8 target glycoproteins and their downstream signaling during breast cancer progression, we used stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative glycoproteomics. We identified glycoproteins differentially expressed in core fucose-specific lectin Lens culinaris agglutinin (LCA)-enriched fractions from aggressive breast cancer cells and compared these to their FUT8-KO counterparts. We first established FUT8-KO cell lines from two highly invasive breast cancer cell lines, MDA-MB-231 and Hs578T, by using the CRISPR-Cas9 system [18]. We selected two independent CRISPR sites targeting exons 3 and 6 of FUT8 ( Fig. 1). After transfecting the FUT8-targeting CRISPR/ Cas9 plasmids into MDA-MB-231 ( Fig. 1a) or Hs578T ( Fig. 1b) cells, individual FUT8-kncockout cell clones were isolated by cell sorting, and the CRISPR/Cas9mediated insertion or deletion mutations were verified at both genomic DNA and mRNA levels ( Fig. 1a, b, upper panel), as was the protein level by western blot analysis ( Fig. 1a, b, lower-left panel) and the consequent loss of core fucosylation at the cell surface by LCA binding assay ( Fig. 1a, b, lower-right panel).
Cell migration and invasion are the critical steps in tumor progression and metastasis. In agreement with our previous report [11], genetic ablation of FUT8 significantly reduced the migratory and invasive ability of MDA-MB-231 (Fig. 1c, e) and Hs578T cells (Fig. 1d, f ), respectively.

Identification and functional signaling pathway analyses of FUT8 targets
To identify novel FUT8 target glycoproteins and their signaling pathways involved in breast cancer cell invasiveness, we used SILAC-based quantitative proteomic analyses to measure the differences in the core-fucosylated proteins between parental and FUT8-KO MDA-MB-231 or Hs578T cells. Parental MDA-MB-231 and Hs578T cells were cultured in 'light' SILAC media, whereas two independent FUT8-KO clones of each cell lines were grown in 'medium' SILAC media or in 'heavy' SILAC media (Fig. 2a). Cell lysates from the three SILAC-labeled cell populations were mixed in equal amounts. Core-fucosylated proteins were enriched by using fucose-specific LCA agarose beads. In line with the common approach for SILAC analysis, we set the threshold for downregulated proteins at 2.0-fold (SILAC ratio < 0.5). After removing apparent contaminated proteins (known to lack consensus N-linked glycosites), we reproducibly identified 282 or 250 target glycoproteins expressed in the invasive breast cancer MDA-MB-231 or Hs578T cells, respectively. We focused further on 140 core fucosylated proteins common to the two lines ( Fig. 2b and Additional file 1: Table S1). Of note, we identified > 98% or 93% of FUT8 targets in two independent KO clones derived from these two lines, which demonstrates the sensitivity and reproducibility of our quantitative proteomics (SILAC) method.
To investigate the most enriched biological functions and molecular networks of FUT8 targets in invasive breast cancer cells, we used disease and function analysis of the two sets of FUT8 target glycoproteins with QIA-GEN Ingenuity Pathway Analysis (IPA) software. Of note, the top 10 categories of molecular and cellular functions identified by these FUT8 targets from the two breast cancer cell lines seemed completely identical although in slightly different order (Additional file 2: Fig. S1a and  b), which suggests that FUT8 indeed acts as a key regulator driving similar downstream pathologic processes of breast cancer (Additional file 2: Fig. S1c). These enriched pathways include cell-to-cell signaling and interaction, cellular movement, cellular growth and proliferation, cell death and survival, cell morphology, cellular assembly and organization (Additional file 2: Fig. S1a and b); the pathways modulate cell motility and cell remodeling and thus regulate cell migration and invasion. These pathways also play an important role in tumorigenesis and cancer metastasis. Another intriguing finding was that the FUT8 target glycoproteins associated with any given functional category were not necessarily identical between these two breast cancer cell lines, yet they are involved in the same molecular and cellular functions (Additional file 3: Fig. S2a-j). These results suggest that upregulated FUT8 plays an important regulatory role during breast cancer progression, which leads to core fucosylation on N-glycans of a variety of membrane proteins, thereby affecting cellular adhesion, migration, receptor signaling function, or interaction with other cells and substrates in a highly complex but coordinated manner.
In addition, we used regulator effects analysis of the common 140 FUT8 target glycoproteins to predict how FUT8 might increase or decrease phenotypic or functional outcomes downstream. Upstream regulator analysis is a novel IPA functional tool that can be used to analyze connections to target proteins via coordinator expression, identifying potential upstream regulators including transcription factors and any gene or small molecule that was observed experimentally to affect gene expression directly or indirectly [19]. Apart from the transcription factors including synoviolin 1 (SYVN1), transcription activator BRG1 (BRG1A), progesterone receptor (PGR), and SAM pointed domain-containing ETS transcription factor (SPDEF), TGF-β1 was also predicted to be an upstream regulator (Fig. 2c). This finding globally agrees with our previous report showing a positive feed-forward mechanism of FUT8-mediated receptor core fucosylation that enhances TGF-β signaling and the EMT, thus stimulating breast cancer cell invasion and metastasis [11]. A similar approach, regulator effect network analysis in IPA, was also used to generate connectivity networks predicting downstream functional effects and phenotypes [19]. The enriched functional networks produced included cell viability and cell proliferation of breast cancer cell lines as well as adhesion, cell movement and chemotaxis migration of many other types of cancer cells including leukemic, lung, prostate, or colorectal cancer cells (Fig. 2c).
To further explore the pathological implications and potential prognostic values, these 140 FUT8 targets were further assessed by using an interactive openaccess database, The Human Protein Atlas database (http:// www. prote inatl as. org). The database contains information about protein localization in normal organs as well as detection of these proteins in 17 different cancer types. Correlation analyses based on their expression in cancer tissue and clinical outcome for cancer patients revealed that many (109/140, 78%) of the FUT8 targets had clinical prognostic values for breast cancer and also other cancer types: cervical, colorectal, endometrial, glioma, head and neck, lung, liver, ovarian, pancreatic, renal, stomach, thyroid, and urothelial cancers ( Fig. 3a and Additional file 4: Table S2). For instance, the Kaplan-Meier survival analysis revealed that breast cancer patients with high expression of ANO6 (Anoctamin 6, a multi-pass transmembrane protein that is a regulator of phospholipid scrambling) [20] or SCARB2 (Scavenger receptor class B member 2, a type III glycoprotein that is located primarily in limiting membranes of lysosomes/endosomes and participates in membrane transportation and the reorganization of endosomal/lysosomal compartment) [15] had a shortened survival compared to those in the low expression group (Fig. 3b, c). Together, these results further verify that the FUT8 target glycoproteins we identified indeed have broader pathological involvement in cancer types beyond breast cancer.

Validation of candidate FUT8 target proteins
From the 140 common FUT8 glycoproteins, we further selected 19 membrane or receptor proteins with potential biological functions in cancer cell growth, proliferation, adhesion, or migration for further validation, including integrins (ITGA2, ITGA3, ITGA5,  ITGA6, ITGAV, ITGB1, and ITGB5), cell-surface proteins (PTK7, TNFRSF21, JAG1, THBD, and CD44) and signaling receptors (AXL, CDCP1, NPR1, PTPRK, EGFR, OSMR, and IL6ST) (Additional file 5: Fig. S3,  Figs. 5a, b and 8a). We first examined whether these targets are truly modified by FUT8-catalyzed core fucosylation by using core fucose-specific LCA lectin blot analysis. When overexpressed in control HEK-293 T cells, all FUT8 candidates were indeed corefucosylated and detected by LCA blotting, which was completely abolished in FUT8-deficient HEK-293 T cell lines (Additional file 5: Fig. S3, Figs. 5a, b and 8a). Importantly, we also performed a negative control by using another lectin RCA I (Ricinus communis agglutinin I) that recognizes terminal galactose or WGA (Wheat germ agglutinin) that binds oligosaccharides containing terminal N-acetylglucosamine. While the FLAG-tagged IL6ST is not core fucosylated (determined by LCA lectin) by FUT8, addition of the terminal galactose (detected by RCA I lectin) or N-acetylglucosamine (detected by WGA) is still glycosylated in the FUT8-KO cells (Additional file 6: Fig. S4). In agreement with these findings, we further confirmed 12 of these candidate FUT8 target proteins including IL6ST, OSMR, ITGA6, ITGAV, ITGB5, AXL, JAG1, NRP1, PTK7, PTPRK, CD44, or TNFRSF21 that are truly core fucosylated in breast carcinoma MDA-MB-231 cells (Fig. 4). Thus, these results validate our overall quantitative glycoproteomics approach in identifying genuine FUT8 targets and also verify that these glycoproteins are authentically modified by core fucosylation.

Functional characterization of novel FUT8 core-fucosylated proteins conferring pro-invasive/pro-metastatic behavior to breast cancer cells
To investigate the roles of core fucosylation of the newly identified FUT8 target proteins, we selected IL6ST and integrin αvβ5 for further study. The cytokine receptor IL6ST is the common signaling subunit for the interleukin (IL)-6 family of cytokines including IL-6, OSM, leukemia inhibitory factor, cardiotrophin-1, and ciliary neurotrophic factor. Binding of IL-6 to IL-6 receptor (IL-6R) induces homodimerization and recruitment of IL6ST, which activates downstream signaling [21]. OSM activates a complex of OSM receptor (OSMR) and IL6ST. Numerous studies have reported that IL6ST plays a role in tumor progression and metastasis [22][23][24]. Previous study showed that IL-6 acts as an inducer of an EMT phenotype in breast cancer cells, which implicates its potential to promote breast cancer metastasis [25]. In addition, OSM can promote an EMT/cancer stem celllike phenotype in breast cancer and enhance migration [26]. When overexpressed in control MDA-MB-231 or HEK-293 T cells, IL6ST and OSMR proteins were corefucosylated, but their core fucosylation was completely absent in FUT8-KO counterpart cell lines (Figs. 4 and 5a, b). To further identify the detailed glycan structure, we analyzed the digested glycopeptides of purified IL6ST expressed in HEK-293 T cells by LC-MS/MS. We identified glycopeptides containing N83, N131, N157, N227, N379, N390, and N564 carrying core fucose ( Fig. 5c and Additional file 7: Table S3), but this core fucosylation was not observed in protein samples from FUT8-KO cells (data not shown). We further determined whether ablation of IL6ST core fucosylation could affect the IL-6 signaling pathway. To this end, we evaluated whether FUT8 KO affects IL-6-stimulated transcriptional activity in IL-6 sensor cells, HEK-Blue ™ IL-6 cells, which were generated by stable transfection of HEK-293 cells with the human IL-6R gene and a STAT3-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene [27]. Upon IL-6 stimulation, HEK-Blue ™ IL-6 cells activated STAT3 and secretion of SEAP (Fig. 5d). Levels of STAT3-induced SEAP can be readily monitored by using QUANTI-Blue ™ . FUT8-KO cells showed diminished transcriptional activity induced by IL-6 or OSM. Moreover, IL-6 or OSM-activated STAT3, quantified by its phosphorylation, was significantly reduced in FUT8-KO MDA-MB-231 cells as compared with control cells (Fig. 5e). Next, we sought to determine whether core fucosylation of IL6ST is critical for its pro-invasive ability. We first established IL6T-KO cell lines from MDA-MB-231 (Fig. 6a) and Hs578T (Fig. 6b) by using the CRISPR-Cas9 approach. Depletion of IL6ST significantly reduced the migratory and invasive ability of MDA-MB-231 (Fig. 6c, e) and Hs578T cells (Fig. 6d, f ), respectively. We then generated a mutant form IL6ST-7NQ (substitution of asparagines of identified core fucosylation site N83, N131, N157, N227, N379, N390, and N564 with glutamines; Fig. 7a). Using LCA blotting, we confirmed the loss of core fucosylation in IL6ST-7NQ mutants (Fig. 7b). IL6ST or IL6ST-7NQ mutant were reconstituted back in IL6ST-KO MDA-MB-231 or Hs578T cells to determine whether core fucosylation of IL6ST affect cell migration and invasion, respectively. While re-expression of wildtype IL6ST can rescue the migratory and invasive ability of IL6ST-KO MDA-MB-231 (Fig. 7c, e) and Hs578T cells (Fig. 7d, f ), IL6ST-7NQ mutant fails to restore migration and invasion of IL6ST-KO cells. Together, these results suggest that core fucosylation of IL6ST is critical for its ability to transmit pro-invasive and pro-metastatic IL-6 or OSM signaling. Other FUT8 target proteins that drew our attention were integrins. Integrins are a family of 24 transmembrane heterodimers generated from a combination of 18 α integrin and 8 β integrin subunits. Much literature has reported the roles of integrins in human cancer progression [28,29]. Altered integrin expression during breast cancer progression has been reported in several studies [30,31]. Apart from their expression, the cellular function of integrins could also be regulated at post-translational levels. For example, core fucosylation is required for the function of integrin α3β1-mediated cell migration and interaction of α4β1 integrin and vascular cell adhesion molecule 1 [13,32]. Nevertheless, the impact of core fucosylation on other integrins during breast cancer progression remains largely unknown. The introduction of dominant-negative E-cadherin increased migration on vitronectin due to increased activity of αvβ5 integrins [33]. Integrin αvβ5 has a role in cell invasion. Some studies suggested that αvβ5-dependent breast cancer migration may be partly regulated by urokinase [34]. Therefore, we further focused on integrin αvβ5 and validated that integrins αv or β5 subunit was indeed core-fucosylated by LCA blotting (Figs. 4 and 8a). In addition, glycan profiling of purified αv or β5 subunit identified glycopeptides containing N-glycosylation sites at N74, N554, N615, N704, N874 of integrin αv. Sites at N347, N 477, N552, N586, N654 of integrin β5 were verified as carrying core fucose (Fig. 8b, Additional file 7: Table S3 and Additional file 8: Fig. S5). Core fucosylation of these sites was completely abolished in samples from FUT8-KO cells (data not shown).
Integrins bind to extracellular matrix components and provide the traction necessary for cell motility and invasion that are critical for cancer progression and metastasis. For example, the interaction of α3β1 integrin with the ligand laminin-5 promotes the migration and invasion of malignant glioma and melanoma cells [35]. The vitronectin-binding receptor αvβ5 integrin has a crucial role in breast malignancy [36]. Metastatic breast cancer cells showed increased expression of αvβ5 integrin. In addition, αvβ5 integrin promotes cancer metastasis via activation of extracellular-signal-regulated kinase 5 [37]. We further investigated a functional combination of integrins in MDA-MB-231 cells by using function-blocking monoclonal antibodies against αvβ5 or αv integrin. We also included function-blocking monoclonal antibodies against β1 integrins because core fucosylation is essential for the functions of α3β1 integrin [13]. The antibody against αvβ5, an αv integrin, almost completely abolished MDA-MB-231 adhesion to vitronectin but had no effect on laminin-5 binding; whereas anti-β1 integrin antibodies were more effective at preventing cell adhesion to laminin-5 (Fig. 8c, d). Thus, αvβ5 integrin likely mediates cell adhesion to vitronectin, whereas β1 likely binds to both vitronectin and laminin-5 in MDA-MB-231 cells. Next, we evaluated the adhesive ability of parental or FUT8-KO MDA-MB-231 cells on the integrin ligands vitronectin and laminin-5. As compared with parental MDA-MB-231 cells, FUT8-KO cells showed significantly reduced adhesion to vitronectin and laminin-5 (Fig. 8e,  f ). Collectively, these results suggest that αvβ5 integrin is a key molecule for cell adhesion on vitronectin in MDA-MB-231 cells and that core fucosylation probably regulates αvβ5 integrin-mediated cell adhesion.

Discussion
To identify novel targets for improving new diagnostic approaches and therapeutic strategies for breast cancer, one must have a more complete understanding of breast cancer biology at the molecular level. We recently identified FUT8 as a key regulator during breast cancer metastasis [11]. However, the full spectrum of FUT8 target glycoproteins and a comprehensive network of signaling pathways orchestrated by FUT8 for breast cancer cell migration and invasion leading to metastasis remain elusive and uninvestigated. In the present study, we Hs578T breast cancer cells to reveal a broad list of core fucosylated target glycoproteins. Our SILAC-based comparative quantitative glycoproteomics analysis identified core-fucosylated proteins, with enrichment in membrane glycoproteins involved in cancer cell migration and invasion. The FUT8 target glycoproteins in breast cancer we identified in general agree with those in previous studies of melanoma or non-small cell lung cancer by sharing greater than 78% coverage [14,16]. Therefore, these findings further validated the effectiveness of identifying FUT8 targets by our SILAC-based quantitative glycoproteomics approach. Some of these FUT8 target proteins have been reported in other human cancers. For example, EGFR and MET core fucosylation can potentiate their ligand binding ability [12,38] and thus may enhance downstream signaling to support tumor growth and metastasis in liver cancer. Integrins are critical for metastasis [39]. Deletion of Fut8 significantly impaired integrin α3β1-mediated cell migration of mouse embryonic fibroblasts [13]. The receptor tyrosine kinase AXL has been implicated in EMT, invasion and chemoresistance of several human cancers [40]. Moreover, aberrant glycosylation of AXL was reported to mediate tumor cell proliferation, invasion and metastasis [41]. Neuropilin-1 is a non-tyrosine kinase receptor implicated in tumor progression [42]. Growing evidence implicates a role for neuropilin-1 in promoting cancer progression by acting as a co-receptor for molecules involved in the EMT pathway and metastasis in tumor tissue such as vascular endothelial growth factor, placental growth factor, and TGF-β1 [43].
Importantly, we further verified the specific core-fucosylation sites and potential glycan composition of each site in the signaling receptor IL6ST and adhesion receptor αvβ5. We demonstrated that core fucosylation of IL6ST promoted IL-6-and OSM-stimulated cellular signaling important for breast cancer EMT and metastasis. It also promoted αvβ5 integrin-mediated cell adhesion on vitronectin in aggressive breast cancer cells. Although the precise molecular and structural basis of how core fucosylation modulates biological activities of their targets is not yet fully defined, core fucosylation of IL6ST or integrin αv/ β5 subunits or even any specific residue may contribute to certain conformations optimal for receptor-ligand interactions. These conformations may facilitate signaling activity or enhance affinity between integrin and extracellular substrates. In support of this notion, a recent study used in silico modeling to demonstrate that fucosylated glycan showed more restricted movement than non-fucosylated glycan on the surface of integrin β1, possibly favoring the interaction of glycan with other molecules [44]. Nevertheless, additional investigations are required to elucidate the molecular mechanism underlying the structural and functional consequences of core fucosylation of IL6ST or αvβ5 during the EMT.
Given the critical roles of glycosylation alterations in cancer biology, strategies that reveal aberrant glycosylation of cancer cells would benefit the diagnosis and treatment of cancer. We and others have reported the association of core fucosylation with tumor progression in breast, melanoma, liver, prostate and lung cancers [11,14,16,17,38]. To date, no specific FUT8 inhibitor for core fucosylation is available. However, this inhibitor could result in severe side effects due to vital roles of core fucosylation during developmental and physiological processes. Indeed, genetic inactivation of Fut8 led to emphysema, severe growth retardation and early postnatal lethality in mice [45]. Therefore, better therapeutic approaches may be those aimed at critical FUT8 targets and/or their downstream signaling pathways. Of note, most (> 90%) of FUT8 targets in invasive breast cancer cells are indeed druggable because they are secreted or membrane-anchored glycoproteins. For instance, to limit the above-mentioned off-target side effects, neutralizing antibodies could be developed against aberrant core fucosylation of pro-invasive/pro-metastatic glycoproteins such as integrin αvβ5 or IL6ST identified in this study or cancer growth-promoting cell surface epidermal growth factor receptor [16]. By discriminating FUT8-mediated normal physiological from pathological signaling, these might be more effective therapeutic means.